The mu and delta opioid receptors (MOR, DOR) modulate many of the same brain processes in vivo, including tolerance and anti-nociception in response to opioid drugs. Many groups have found that inhibiting the DOR through various means decreases the side effects of MOR agonists like morphine. While the basis for this interaction is unknown, one strong possibility is the formation of a MOR-DOR heterodimer (MDOR). Many groups have shown that the MDOR can form in heterologous expression systems in vitro, with a unique pharmacology and signal transduction profile when compared to the monomeric forms. Devi and colleagues recently developed an MDOR selective antibody, and used this antibody to demonstrate MDOR upregulation in the brains of mice chronically treated with morphine. Other experiments suggested that the MDOR promotes tolerance, dependence, and drug seeking in vivo. While MDOR selective agonists have been developed, no known drug-like antagonist has ever been created to our knowledge, limiting our ability to determine the role of MDOR in vivo. To address this lack, we created a novel series of potential selective peptide MDOR antagonists by connecting low affinity MOR (H-Tyr-Pro-Phe-D1Nal-NH2) and moderate affinity DOR (Tyr-Tic- OH) pharmacophores with a variable length (15-42 atom) flexible polyamide spacer. We tested this preliminary series in vitro using radioligand binding and 35S-GTP?S coupling in antagonist mode using MOR, DOR, and MDOR expressing cell lines. We found compelling evidence that our preliminary series selectively targets the MDOR, with a selectivity ratio of ~91 fold for our best compound, the 24 atom spacer length. Building from this initial success, we aim in the current proposal to explore the MDOR structure-activity relationship (SAR) of our compound series, and improve compound potency and selectivity at the MDOR by modulating pharmacophore affinity (increased MOR, decreased DOR) as well as linker rigidity (minimally rigid [gly-gly-pro], moderately rigid [gly-pro]). These studies will be performed in an iterative development process, using the best compound from each series to inform the next series, minimizing compound number. After this SAR development, we will use our most potent and selective compound to begin to test MDOR activity in vivo. This will be accomplished by intracerebroventricular (icv) and intrathecal (it) injection of MDOR antagonist into mice prior to tail-flick anti- nociception evoked by MOR (DAMGO), DOR (DSLET), and MDOR (CYM51010) selective agonists. These studies will demonstrate the selectivity of our compound in vivo. We will also pre-treat mice with MDOR antagonist prior to the induction of tolerance and dependence with morphine to begin to explore the in vivo role of the MDOR in these opioid side effects, which has been suggested by the literature. In the short term, this initial optimized series will provide a useful tool to interrogate the role of the MDOR in vivo. Long term, through modifications to improve drugability (glycosylation, etc.), these compounds may provide the basis for selective MDOR targeted therapeutics to improve the side effect profile of opioid therapy.